Bulk-suppressed and surface-sensitive Raman scattering by transferable plasmonic membranes with irregular slot-shaped nanopores.

Raman spectroscopy enables the non-destructive characterization of chemical composition, crystallinity, defects, or strain in countless materials. However, the Raman response of surfaces or thin films is often weak and obscured by dominant bulk signals. Here we overcome this limitation by placing a transferable porous gold membrane, (PAuM) on the surface of interest. Slot-shaped nanopores in the membrane act as plasmonic antennas and enhance the Raman response of the surface or thin film underneath. Simultaneously, the PAuM suppresses the penetration of the excitation laser into the bulk, efficiently blocking its Raman signal. Using graphene as a model surface, we show that this method increases the surface-to-bulk Raman signal ratio by three orders of magnitude. We find that 90% of the Raman enhancement occurs within the top 2.5 nm of the material, demonstrating truly surface-sensitive Raman scattering. To validate our approach, we quantify the strain in a 12.5 nm thin Silicon film and analyze the surface of a LaNiO3 thin film. We observe a Raman mode splitting for the LaNiO3 surface-layer, which is spectroscopic evidence that the surface structure differs from the bulk. These results validate that PAuM gives direct access to Raman signatures of thin films and surfaces.

A proteome scale study reveals how plastic surfaces and agitation promote protein aggregation.

Protein aggregation in biotherapeutics can reduce their activity and effectiveness. It may also promote immune reactions responsible for severe adverse effects. The impact of plastic materials on protein destabilization is not totally understood. Here, we propose to deconvolve the effects of material surface, air/liquid interface, and agitation to decipher their respective role in protein destabilization and aggregation. We analyzed the effect of polypropylene, TEFLON, glass and LOBIND surfaces on the stability of purified proteins (bovine serum albumin, hemoglobin and α-synuclein) and on a cell extract composed of 6000 soluble proteins during agitation (P = 0.1-1.2 W/kg). Proteomic analysis revealed that chaperonins, intrinsically disordered proteins and ribosomes were more sensitive to the combined effects of material surfaces and agitation while small metabolic oligomers could be protected in the same conditions. Protein loss observations coupled to Raman microscopy, dynamic light scattering and proteomic allowed us to propose a mechanistic model of protein destabilization by plastics. Our results suggest that protein loss is not primarily due to the nucleation of small aggregates in solution, but to the destabilization of proteins exposed to material surfaces and their subsequent aggregation at the sheared air/liquid interface, an effect that cannot be prevented by using LOBIND tubes. A guidance can be established on how to minimize these adverse effects. Remove one of the components of this combined stress - material, air (even partially), or agitation - and proteins will be preserved.

Design of tethered bilayer lipid membranes, using wet chemistry via aryldiazonium sulfonic acid spontaneous grafting on silicon and chrome.

We describe a bottom-up surface functionalization to design hybrid molecular coatings that tether biomembranes using wet chemistry. First, a monolayer was formed by immersion in a NH2-Ar-SO3H solution, allowing aryldiazonium salt radicals to spontaneously bind to it via strong C bonding. After formation of the air-stable and dense molecular monolayer (-Ar-SO3H), a subsequent activation was used to form highly reactive -Ar-SO2Cl groups nearly perpendicular to the monolayer. These can bind commercial surfactants, PEGylated oligomers and other inexpensive molecules via their -OH, -COOH, or -NH2 chain end-moieties, to build hybrid coatings. Metal and oxidized chromium, semi-conductor n-doped silicon (111), are the substrates tested for this protocol and the aromatic organic monolayers formed at their surface are characterized by X-ray photoelectron spectroscopy (XPS). XPS reveals unambiguously the presence of C-Cr and C-Si bonds, ensuring robustness of the coatings. Functional sulfur groups (-SO3H) cover up to 6.5×10-10 mol cm-2 of the silicon interface and 4.7×10-10 mol cm-2 of the oxidized chromium interface. These surface concentrations are comparable to the classic values obtained when the prefunctionalisation is driven by electrochemistry on conductors. Tethered lipid membranes formed on these coatings were analyzed by neutron reflectivity at the interface of functionalized n-doped silicon substrates after immersion in a solution of lipid vesicles and subsequent fusion. Results indicate a rather compact hybrid coating of Brij anchor-harpoon molecules that maintain a single lipid bilayer above the substrate, on top of a hydrated PEO cushion.

Semantic confusion regarding the development of multisensory integration: a practical solution.

There is now a good deal of data from neurophysiological studies in animals and behavioral studies in human infants regarding the development of multisensory processing capabilities. Although the conclusions drawn from these different datasets sometimes appear to conflict, many of the differences are due to the use of different terms to mean the same thing and, more problematic, the use of similar terms to mean different things. Semantic issues are pervasive in the field and complicate communication among groups using different methods to study similar issues. Achieving clarity of communication among different investigative groups is essential for each to make full use of the findings of others, and an important step in this direction is to identify areas of semantic confusion. In this way investigators can be encouraged to use terms whose meaning and underlying assumptions are unambiguous because they are commonly accepted. Although this issue is of obvious importance to the large and very rapidly growing number of researchers working on multisensory processes, it is perhaps even more important to the non-cognoscenti. Those who wish to benefit from the scholarship in this field but are unfamiliar with the issues identified here are most likely to be confused by semantic inconsistencies. The current discussion attempts to document some of the more problematic of these, begin a discussion about the nature of the confusion and suggest some possible solutions.

Challenges in quantifying multisensory integration: alternative criteria, models, and inverse effectiveness.

Single-neuron studies provide a foundation for understanding many facets of multisensory integration. These studies have used a variety of criteria for identifying and quantifying multisensory integration. While a number of techniques have been used, an explicit discussion of the assumptions, criteria, and analytical methods traditionally used to define the principles of multisensory integration is lacking. This was not problematic when the field was small, but with rapid growth a number of alternative techniques and models have been introduced, each with its own criteria and sets of implicit assumptions to define and characterize what is thought to be the same phenomenon. The potential for misconception prompted this reexamination of traditional approaches in order to clarify their underlying assumptions and analytic techniques. The objective here is to review and discuss traditional quantitative methods advanced in the study of single-neuron physiology in order to appreciate the process of multisensory integration and its impact.

The development of cortical multisensory integration.

Although there are many perceptual theories that posit particular maturational profiles in higher-order (i.e., cortical) multisensory regions, our knowledge of multisensory development is primarily derived from studies of a midbrain structure, the superior colliculus. Therefore, the present study examined the maturation of multisensory processes in an area of cat association cortex [i.e., the anterior ectosylvian sulcus (AES)] and found that these processes are rudimentary during early postnatal life and develop only gradually thereafter. The AES comprises separate visual, auditory, and somatosensory regions, along with many multisensory neurons at the intervening borders between them. During early life, sensory responsiveness in AES appears in an orderly sequence. Somatosensory neurons are present at 4 weeks of age and are followed by auditory and multisensory (somatosensory-auditory) neurons. Visual neurons and visually responsive multisensory neurons are first seen at 12 weeks of age. The earliest multisensory neurons are strikingly immature, lacking the ability to synthesize the cross-modal information they receive. With postnatal development, multisensory integrative capacity matures. The delayed maturation of multisensory neurons and multisensory integration in AES suggests that the higher-order processes dependent on these circuits appear comparatively late in ontogeny.

Visual experience is necessary for the development of multisensory integration.

Multisensory neurons and their ability to integrate multisensory cues develop gradually in the midbrain [i.e., superior colliculus (SC)]. To examine the possibility that early sensory experiences might play a critical role in these maturational processes, animals were raised in the absence of visual cues. As adults, the SC of these animals were found to contain many multisensory neurons, the large majority of which were visually responsive. Although these neurons responded robustly to each of their cross-modal inputs when presented individually, they were incapable of synthesizing this information. These observations suggest that visual experiences are critical for the SC to develop the ability to integrate multisensory information and lead to the prediction that, in the absence of such experience, animals will be compromised in their sensitivity to cross-modal events.

Non-stationarity in multisensory neurons in the superior colliculus.

The superior colliculus (SC) integrates information from multiple sensory modalities to facilitate the detection and localization of salient events. The efficacy of "multisensory integration" is traditionally measured by comparing the magnitude of the response elicited by a cross-modal stimulus to the responses elicited by its modality-specific component stimuli, and because there is an element of randomness in the system, these calculations are made using response values averaged over multiple stimulus presentations in an experiment. Recent evidence suggests that multisensory integration in the SC is highly plastic and these neurons adapt to specific anomalous stimulus configurations. This raises the question whether such adaptation occurs during an experiment with traditional stimulus configurations; that is, whether the state of the neuron and its integrative principles are the same at the beginning and end of the experiment, or whether they are altered as a consequence of exposure to the testing stimuli even when they are pseudo-randomly interleaved. We find that unisensory and multisensory responses do change during an experiment, and that these changes are predictable. Responses that are initially weak tend to potentiate, responses that are initially strong tend to habituate, and the efficacy of multisensory integration waxes or wanes accordingly during the experiment as predicted by the "principle of inverse effectiveness." These changes are presumed to reflect two competing mechanisms in the SC: potentiation reflects increases in the expectation that a stimulus will occur at a given location relative to others, and habituation reflects decreases in stimulus novelty. These findings indicate plasticity in multisensory integration that allows animals to adapt to rapidly changing environmental events while suggesting important caveats in the interpretation of experimental data: the neuron studied at the beginning of an experiment is not the same at the end of it.

Postnatal experiences influence how the brain integrates information from different senses.

Sensory processing disorder (SPD) is characterized by anomalous reactions to, and integration of, sensory cues. Although the underlying etiology of SPD is unknown, one brain region likely to reflect these sensory and behavioral anomalies is the superior colliculus (SC), a structure involved in the synthesis of information from multiple sensory modalities and the control of overt orientation responses. In the present review we describe normal functional properties of this structure, the manner in which its individual neurons integrate cues from different senses, and the overt SC-mediated behaviors that are believed to manifest this "multisensory integration." Of particular interest here is how SC neurons develop their capacity to engage in multisensory integration during early postnatal life as a consequence of early sensory experience, and the intimate communication between cortex and the midbrain that makes this developmental process possible.

Excitotoxic lesions of the superior colliculus preferentially impact multisensory neurons and multisensory integration.

The superior colliculus (SC) plays an important role in integrating visual, auditory and somatosensory information, and in guiding the orientation of the eyes, ears and head. Previously we have shown that cats with unilateral SC lesions showed a preferential loss of multisensory orientation behaviors for stimuli contralateral to the lesion. Surprisingly, this behavioral loss was seen even under circumstances where the SC lesion was far from complete. To assess the physiological changes induced by these lesions, we employed single unit electrophysiological methods to record from individual neurons in both the intact and damaged SC following behavioral testing in two animals. In the damaged SC of these animals, multisensory neurons were preferentially reduced in incidence, comprising less than 25% of the sensory-responsive population (as compared with 49% on the control side). In those multisensory neurons that remained following the lesion, receptive fields were nearly twofold larger, and less than 25% showed normal patterns of multisensory integration, with those that did being found in areas outside of the lesion. These results strongly suggest that the multisensory behavioral deficits seen following SC lesions are the combined result of a loss of multisensory neurons and a loss of multisensory integration in those neurons that remain.

Visual deprivation alters the development of cortical multisensory integration.

It has recently been demonstrated that the maturation of normal multisensory circuits in the cortex of the cat takes place over an extended period of postnatal life. Such a finding suggests that the sensory experiences received during this time may play an important role in this developmental process. To test the necessity of sensory experience for normal cortical multisensory development, cats were raised in the absence of visual experience from birth until adulthood, effectively precluding all visual and visual-nonvisual multisensory experiences. As adults, semichronic single-unit recording experiments targeting the anterior ectosylvian sulcus (AES), a well-defined multisensory cortical area in the cat, were initiated and continued at weekly intervals in anesthetized animals. Despite having very little impact on the overall sensory representations in AES, dark-rearing had a substantial impact on the integrative capabilities of multisensory AES neurons. A significant increase was seen in the proportion of multisensory neurons that were modulated by, rather than driven by, a second sensory modality. More important, perhaps, there was a dramatic shift in the percentage of these modulated neurons in which the pairing of weakly effective and spatially and temporally coincident stimuli resulted in response depressions. In normally reared animals such combinations typically give rise to robust response enhancements. These results illustrate the important role sensory experience plays in shaping the development of mature multisensory cortical circuits and suggest that dark-rearing shifts the relative balance of excitation and inhibition in these circuits.

On the use of superadditivity as a metric for characterizing multisensory integration in functional neuroimaging studies.

A growing number of brain imaging studies are being undertaken in order to better understand the contributions of multisensory processes to human behavior and perception. Many of these studies are designed on the basis of the physiological findings from single neurons in animal models, which have shown that multisensory neurons have the capacity for integrating their different sensory inputs and give rise to a product that differs significantly from either of the unisensory responses. At certain points these multisensory interactions can be superadditive, resulting in a neural response that exceeds the sum of the unisensory responses. Because of the difficulties inherent in interpreting the results of imaging large neuronal populations, superadditivity has been put forth as a stringent criterion for identifying potential sites of multisensory integration. In the present manuscript we discuss issues related to using the superadditive model in human brain imaging studies, focusing on population responses to multisensory stimuli and the relationship between single neuron measures and functional brain imaging measures. We suggest that the results of brain imaging studies be interpreted with caution in regards to multisensory integration. Future directions for imaging multisensory integration are discussed in light of the ideas presented.

Superior colliculus neurons use distinct operational modes in the integration of multisensory stimuli.

Many neurons in the superior colliculus (SC) integrate sensory information from multiple modalities, giving rise to significant response enhancements. Although enhanced multisensory responses have been shown to depend on the spatial and temporal relationships of the stimuli as well as on their relative effectiveness, these factors alone do not appear sufficient to account for the substantial heterogeneity in the magnitude of the multisensory products that have been observed. Toward this end, the present experiments have revealed that there are substantial differences in the operations used by different multisensory SC neurons to integrate their cross-modal inputs, suggesting that intrinsic differences in these neurons may also play an important deterministic role in multisensory integration. In addition, the integrative operation employed by a given neuron was found to be well correlated with the neuron's dynamic range. In total, four categories of SC neurons were identified based on how their multisensory responses changed relative to the predicted addition of the two unisensory inputs as stimulus effectiveness was altered. Despite the presence of these categories, a general rule was that the most robust multisensory enhancements were seen with combinations of the least effective unisensory stimuli. Together, these results provide a better quantitative picture of the integrative operations performed by multisensory SC neurons and suggest mechanistic differences in the way in which these neurons synthesize cross-modal information.

Neuron-specific response characteristics predict the magnitude of multisensory integration.

Multisensory neurons in the superior colliculus (SC) typically respond to combinations of stimuli from multiple modalities with enhancements and/or depressions in their activity. Although such changes in response have been shown to follow a predictive set of integrative principles, these principles fail to completely account for the full range of interactions seen throughout the SC population. In an effort to better define this variability, we sought to determine if there were additional features of the neuronal response profile that were predictive of the magnitude of the multisensory interaction. To do this, we recorded from 109 visual-auditory SC neurons while systematically manipulating stimulus intensity. Along with the previously described roles of space, time, and stimulus effectiveness, two features of a neuron's response profile were found to offer predictive value as to the magnitude of the multisensory interaction: spontaneous activity and the level of sensory responsiveness. Multisensory neurons with little or no spontaneous activity and weak sensory responses had the capacity to exhibit large response enhancements. Conversely, neurons with modest spontaneous activity and robust sensory responses exhibited relatively small response enhancements. Together, these results provide a better view into multisensory integration, and suggest substantial heterogeneity in the integrative characteristics of the multisensory SC population.